The glucocorticoid steroids regulate a wide variety of functions in the body and synthetic analogs are used in the therapy of many diseases. These hormones act via receptors that regulate gene expression, and understanding how steroids "turn on" their receptors constitutes a fundamental problem in molecular endocrinology. The first step in the pathway that leads to activation of gene transcription is the binding of the steroid to the receptor. It is clear that non-receptor components of cytosol are important for determining the steroid binding configuration of the receptor and this grant focuses on how those cytosolic factors work. Thiol moieties must be in a reduced form for the receptor to bind steroid, for receptor transformation, and for DNA binding. We have shown that all three of these functions are blocked by hydrogen peroxide which promotes disulfide bond formation (Tienrungro) et al. J. Biol. Chem. 262:6992, 1987). We have also shown that the steroid binding capacity of the receptor is regulated by an endogenous cytosolic :educing factor, which has been identified as the thiol disulfide exchange protein thioredoxin (Grippo et al. J. Biol. Chem. 260:93, 1985). Two specific aims of this proposal use direct physical studies of the receptor protein to determine the mechanism of the peroxide effect and the thioredoxin effect. A second endogenous factor is a metal anion that produces the same effects on the receptor as molybdate (Meshinchi et al. submitted to J. Biol. Chem.), which appears to stabilize the receptor by interacting with functional groups located in the steroid binding domain (Pratt et al. J. Biol. Chem., in press). Two specific aims are 1) to identify the endogenous metal anion (or anions) and 2) to identify the specific site of molybdate action via site-specific modification of the receptor protein. Molybdate and the endogenous metal factor stabilize the association of the glucocorticoid receptor with the 90-kDa heat shock protein (Sanchez et al. J. Biol. Chem. 262.6986,1987). This complex is also stabilized by peroxide (Bresnick et al. J. Steroid Biochem., in press), and we have developed evidence that the receptor must be in this heteromeric complex for the appropriate steroid binding conformation. Several specific aims focus on studying the steroid binding activity of the immunopurified heteromeric complex to determine the non-receptor components of cytosol that must be present to form and maintain the receptor in its steroid binding state.